CN109097434B - 一种唾液酸检测试剂盒 - Google Patents
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Abstract
本发明公开了一种唾液酸检测试剂盒，属于临床体外检测试剂技术领域。本发明试剂盒包含试剂R1、R2和校准品。通过在试剂R1中加入由MgCl2、还原型谷胱甘肽、EDTA三种成分组成的酶激活剂，有效的提高了试剂盒的分析灵敏度，其线性范围也较好，试剂的准确度高，稳定性好，有利于在市场中进一步的推广使用。
Description
技术领域
本发明涉及临床体外检测试剂技术领域，特别涉及一种唾液酸检测试剂盒。
背景技术
唾液酸（SA），学名叫作“N-乙酰基神经氨酸”，唾液酸是广泛存在于生物系统中的一类天然糖类化合物，最早由Blix从牛下颌唾液腺粘蛋白中分离得到，所以命名为唾液酸。它是以九碳糖神经氨酸为基本结构的一族衍生物的总称，共有 50 余种，广泛存在于生物体内细胞膜糖蛋白和脂蛋白中，并在生命体许多重要过程中发挥着重要的作用，如参与细胞识别、生存、繁衍、生物膜流动、细胞内吞作用。
纯唾液酸是无色的,易溶于水,在水溶液中不发生变旋作用。纯的一乙酞神经氨酸和一轻乙酞神经氨酸在水溶液中很稳定，4℃时贮藏数月不发生变化。如果溶液中含有非常微量的有机酸,其稳定性就受到很大的影响。二乙酞和三乙酞神经氨酸不稳定,在常温下很易转变成一乙酞神经氨酸。
唾液酸是人类大脑认知发育必要的营养素，在植物中是不存在的，其大量存在于脊椎动物和高等无脊椎动物体内，如羊肉、猪肉及牛肉，鸡蛋中富有唾液酸，母乳中唾液酸的含量远远高于牛乳，而家禽和鱼类含量却很少。有关唾液酸消化和吸收机制鲜为人知，目前的最新研究数据只停留在哺乳动物身上。人体的很多细胞，血细胞、纤维细胞、神经元细胞等，都可以通过胞饮、细胞内吞作用和溶酶体转运吸收外源性游离的唾液酸进入细胞内，被吸收的唾液酸即释放或被细胞内的唾液酸酶所水解，再被转运至胞液中，在细胞核中被激活后，转换成胞苷酸唾液酸 (Cytidine monophosphospate-Sialic acid, CMP-SA)，在高尔基体中形成配糖体。
人血清或血浆中唾液酸参考范围为45.6 mg/dL～75.4mg/dL。人体中唾液酸主要来源于葡萄糖代谢的中间产物，其值一般会由于炎症性疾病及伴随组织破坏而来的疾病而升高。
唾液酸检测试剂盒（神经氨酸苷酶法）检测原理为样品中的唾液酸受神经氨酸苷酶的作用，形成N-乙酰神经氨酸，进而在NANA-醛缩酶的作用下生成丙酮酸和N-乙酰甘露糖醇。丙酮酸在NADH存在下由乳酸脱氢酶（LDH）作用下生成乳酸和NAD+，通过测定NADH吸光度下降速率即可得到样品中唾液酸的浓度。
唾液酸检测试剂盒（神经氨酸苷酶法）是一种无需预处理样本，技术和设备要求不高，而精密度和特异性更高的分析方法，由于该方法不需要昂贵的设备，可以实现自动化，且可测定大量标本，因此受到临床广泛推广。本发明在不改变其它性能指标的前提下加强了试剂的分析灵敏度。
发明内容
本发明提供了一种唾液酸检测试剂盒。该试剂盒与常规的试剂盒相比，准确度、线性范围、稳定性等性能指标一致，而分析灵敏度有所提高，有利于试剂在临床上的推广应用。
本发明是通过以下措施实现的：
一种唾液酸检测试剂盒，其特征在于，它包含试剂R1、R2和校准品，其中试剂R1组成为：
Tris-HCL缓冲液（pH7.0） 0.1mol/L
神经氨酸苷酶 0.5U/mL
乳酸脱氢酶 3U/mL
Proclin300 3ml/L
MgCl2 1- 2g/L
还原型谷胱甘肽 1- 2g/L
EDTA 2-4g/L
试剂R2组成为：
Tris-HCL缓冲液(pH9.0 ) 0.1mol/ L
还原型辅酶Ⅰ(NADH) 0.2mmol/L
N-乙酰神经醛缩酶 4U/mL
Proclin300 3ml/L。
所述样本和试剂R在使用时的体积比例为体积比例为S：R1：R2=7µL：210µL：70µL。
本实施例描述的唾液酸检测试剂盒（神经氨酸苷酶法），在使用时，其测定方法是采用自动生化分析仪，如日立7180全自动分析仪，操作方法：加入生理盐水、样本或校准品7μl，之后再加入试剂1为210μl预孵育5min ，加入试剂2为70μl，3分钟后开始记录吸光度值A1，连续监测90秒，读取吸光度A2，计算ΔA/min。
本发明所使用的校准品为英国朗道公司生产的复合校准品。
本发明的有益效果：
本发明提供的唾液酸检测试剂盒，通过在试剂中加入了由MgCl2、还原型谷胱甘肽、EDTA三种成分组成的酶激活剂，各组分协同作用提高了酶的活性，从而提高了试剂盒的分析灵敏度，却不会对试剂的准确度、线性和稳定性产生影响，有利于该试剂在市场中进一步的推广。
附图说明
图1 实施例1准确度验证实验检测结果与对比例1检测结果相关性；
图2 实施例2准确度验证实验检测结果与对比例1检测结果相关性；
图3 线性相关性验证实验检测结果；
图4 稳定性验证实验检测结果；
图5 分析灵敏度验证实验结果。
具体实施方式
为了更好的理解本发明，下面结合具体对比例与实施例来进一步详细说明。
对比例1
市场上获得认可的一种准确度优异的唾液酸试剂盒。
试剂R1组成为：
Tris-HCL缓冲液（pH7.0） 0.1mol/L
神经氨酸苷酶 0.5U/mL
乳酸脱氢酶 3U/mL
Proclin300 3ml/L
试剂R2组成为：
Tris-HCL缓冲液(pH9.0 ) 0.1mol/ L
还原型辅酶Ⅰ(NADH) 0.2mmol/L
N-乙酰神经醛缩酶 4U/mL
Proclin300 3ml/L。
本实施例描述的试剂盒，在使用时，其测定方法是采用自动生化分析仪，如日立7180自动分析仪，操作如下：
加入生理盐水、样本或校准品7μl，之后再加入试剂1为210μl预孵育5min ，加入试剂2为70μl，3分钟后开始记录吸光度值A1，连续监测90秒，读取吸光度A2，计算ΔA/min。
本实施例所使用的校准品为英国朗道公司生产的复合校准品。
实施例1
一种唾液酸检测试剂盒。
试剂R1组成为：
Tris-HCL缓冲液（pH7.0） 0.1mol/L
神经氨酸苷酶 0.5U/mL
乳酸脱氢酶 3U/mL
Proclin300 3ml/L
MgCl2 1g/L
还原型谷胱甘肽 1g/L
EDTA 2g/L
试剂R2组成为：
Tris-HCL缓冲液(pH9.0 ) 0.1mol/ L
还原型辅酶Ⅰ(NADH) 0.2mmol/L
N-乙酰神经醛缩酶 4U/mL
Proclin300 3ml/L。
具体测定方法同对比例1。
实施例2
一种唾液酸检测试剂盒。
试剂R1组成为：
Tris-HCL缓冲液（pH7.0） 0.1mol/L
神经氨酸苷酶 0.5U/mL
乳酸脱氢酶 3U/mL
Proclin300 3ml/L
MgCl2 2g/L
还原型谷胱甘肽 2g/L
EDTA 4g/L
试剂R2组成为：
Tris-HCL缓冲液(pH9.0 ) 0.1mol/ L
还原型辅酶Ⅰ(NADH) 0.2mmol/L
N-乙酰神经醛缩酶 4U/mL
Proclin300 3ml/L。
具体测定方法同对比例1。
对上述对比例和实施例中制得的试剂盒分析性能进行实验验证。
准确度验证实验：
将实施例1、2的试剂盒作为实验组，对比例1试剂盒作为对照组进行对比实验，对40个样本进行检测，检测的结果如图1、图2。
通过图1、图2的检测数据可知，实施例1、2试剂盒与对比例1试剂盒的检测结果线性相关系数r分别为0.9983、0.9982，相关性比较好，表明本发明的试剂盒与市场上获得认可的具有优异准确度的唾液酸检测试剂盒有高度一致性，证明本发明试剂盒添加的其他各种成分对其准确性不会造成影响，试剂盒依然保持较好的准确度。
线性相关性验证实验：
找到唾液酸高值样本为200mg/dL，用生理盐水进行系列稀释，配制6个不同浓度的样本，依次为200 mg/dL、160 mg/dL、120 mg/dL、80 mg/dL、40mg/dL、0 mg/dL浓度的样本，每个浓度水平各样本分别测定三次，分别取其平均值。分别利用对比例1和实施例1、2的试剂进行检测。检测结果见图3。
上述检测结果显示，对比例1和实施例1、2检测结果相关性均大于0.990，达到产品标准要求，说明在试剂中加入酶激活剂不会降低试剂检测的线性相关性。
稳定性验证实验：
在2℃～8℃、无腐蚀性气体的避光环境中贮存试剂，检测三种试剂的稳定性。三种试剂每月选取同一样本测定三次，取平均值，与新鲜的对比例1试剂检测结果进行比对，从而确定试剂的稳定时间。检测数据见图4。
实验结果显示，三组试剂在2℃～8℃、无腐蚀性气体的避光环境中贮存15个月稳定，证明本发明试剂盒添加的其他各种成分对其稳定性不会造成影响，试剂盒依然保持较好的稳定性。
分析灵敏度验证实验：
用唾液酸试剂（盒）测试已知浓度在1000 mg/dL的样本，记录吸光度变化率（ΔA/min）。分别利用对比例1、实施例1、2的试剂进行检测，检测结果见图5。
通过检测数据可知，实施例1、2检测试剂盒的吸光度差值均比对比例1的高，说明在试剂中加入由MgCl2、还原型谷胱甘肽、EDTA三种成分组成的酶激活剂能有效的提高唾液酸检测试剂盒的分析灵敏度。
综合以上分析，本发明提供的唾液酸检测试剂盒，在试剂R1中通过加入酶激活剂能够有效的提高试剂盒的分析灵敏度，线性范围较好，试剂的准确度和稳定性也较好。因此，本发明提供的唾液酸检测试剂盒有利于在市场中进一步的推广使用。
Claims (2)
1.一种唾液酸测定试剂盒，其特征在于，它包含试剂R1、R2和校准品，其中试剂R1组成为：
试剂R2组成为：
2.根据权利要求1所述的试剂盒，其特征在于，所述样本和试剂R1、R2在使用时的体积比例为S：R1：R2＝7μL：210μL：70μL。
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